Prog. Neuro-Psychophormaco1. C Biol. Psychiat. 1987, Vol. 11, pp. 683-699 0278-5846187 $0.00+.50

Printed in Great Britain. All rights reserved. Copyright 0 1987 Pergamon 1ournals Ltd.

PLATELET MEMBRANE FLUIDITY IDENTIFIES A CLINICAL SUBTYPE OF ALZHEIMER’S DISEASE

GEORGE S. ZUBENK01p3y4,, BRUCE M. COHEN3, CHARLES F. REYNOLDS, IIIl, FRANCOIS BOLLERlp2,

IVANA TEPLY l, BONNIE CHOJNACK14

1Departments of Psychiatry and 2Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 3Mailman Research Center, McLean Hospital, Belmont, MA, and 3Department of

Psychiatry, Harvard Medical School, Boston, MA; and 4Department of Biological Sciences, Mellon Institute, Carnegie Mellon University, Pittsburgh, PA

(Final form, June 1987)

Abstracl

Zubenko, George S., Bruce M. Cohen, Charles F. Reynolds, III, Francois Boller, Ivana Teply, Bonnie Chojnacki: Platelet membrane fluidity identifies clinical subtype of Alzheimer’s disease. Prog. Neuropsychopharmacol. & Biol. Psychiat. 1987, 11: 683 -699. -

1. The fluorescence anisotropy of 1,6-diphenyl-1,3,5hexatriene (DPH) in labeled platelet membranes, an index of membrane fluidity, identifies a prominent subgroup (approx. 50%) of patients with Alzheimer’s disease who manifest distinct clinical features.

2. We review an integrated series of studies that explore both the clinical significance of this fmding and the biological basis for the platelet membrane alteration.

Alzheimer’s disease, biological/clinical subtype, platelet membranes Keywords:

Abbreviatiom 1,6-diphenyl-1,3,5hexatriene (DPH), phosphate buffered saline (PBS), 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMADPH)

Introduction

Mounting evidence suggests that Alzheimer’s disease is associated with pathologic changes in cells

outside the central nervous system (for reviews see Blass and Zemcov 1984, Hollander et al 1986).

Several of the abnormalities described in nonneural cells reflect an alteration in cell membrane structure or

function. Among these is our initial report of an increase in platelet membrane fluidity, as revealed by a

decrease in the fluorescence anisotropy of 1,6 diphenyl-1,3,5hexatriene (DPH) in labeled membranes

(Zubenko et al 1984, Zubenko and Cohen 1985a). Abnormalities of cell membrane composition and

structure have also been found in brain tissue obtained at autopsy from patients who died with confirmed

Alzheimer’s disease. These include changes in brain phospholipid metabolism as revealed by 3lP-NMR

spectroscopy (Pettegrew et al 1984, Barany et al 1985), disordering of cortical myelin as indicated by X-

683

664 G. S. Zubenko et al

ray diffraction (Chia et al 1984), and an alteration in the molecular dynamics of hippocampal membranes

as reflected by fluorescence spectroscopy (Zubenko 1986).

It is difficult to study brain disease and currently impossible to establish the diagnosis of Alzheimer’s

disease without the examination of brain tissue. Given these problems, the impact of a reliable and specific

antemortem, peripheral tissue marker for Alzheimer’s disease would be substantial. Such a marker would

not only serve as a clue to the biological basis of Alzheimer’s disease, but by allowing accurate diagnosis

during life, would improve clinical research on the epidemiology and treatment of Alzheimer’s disease as

well. The reliability of the finding of abnormal platelet membrane fluidity in Alzheimer’s disease is already

suggested by its replicability in our studies and those of other investigators (Hicks et al 1987). In this

manuscript, we review an interrelated series of studies that confirm our initial observation of increased

platelet membrane fluidity in Alzheimer’s disease, : ;sess the specificity of this finding, examine the

clinical correlates of increased platelet membrane fluidity in patients with Alzheimer’s disease, and explore

the biological basis for this platelet membrane alteration (Zubenko and Cohen 1985a, Cohen et al 1987,

Zubenko et al 1987a-d).

Methods

The study population consisted of 125 subjects who were at least 45 years of age. A complete history

and physical examination, including detailed neurologic, psychiatric, and mental status examinations, were

performed for each of the 125 subjects. All subjects had a normal complete blood count, urinalysis, blood

chemistry screen, serum folate and B12 levels, and thyroid function tests, and none were suffering from

malnutrition or vitamin deficiency syndromes as determined by clinical and laboratory evaluations.

Subjects with disorders that affect blood cell membrane lipid composition or serum lipid profiles,

including alcohol or other substance abuse, generalized atherosclerosis, familial lipidoses, diabetes

mellitus, uremia, and nephrosis, as well as subjects who were on restrictive diets, were excluded from the

study (Shinitzky and Barenholz 1978, Baccus 1976, Hunt 1985).

The normal control group consisted of 50 neurologically-healthy volunteers. The Alzheimer’s disease

group consisted of 5 1 outpatients with “probable” Alzheimer’s disease as defined by currently accepted

clinical criteria (McKhann et al 1984). Individuals who had any history of treatment with neuroleptic or

antidepressant drugs, or were currently being treated with any medication that can affect platelet membrane

fluidity by in vitro or in ViVQ exposure, were excluded from these two groups (Zubenko and Cohen

1985a-c, 1986). In addition, all patients remained free of any drug ingestion, including the use of aspirin,

for at least 24h prior to blood drawing. Forty-eight of the 50 controls and 44 of the 5 1 demented patients

were receiving no prescribed medications. The medications taken by the remaining subjects at the time of

entry into the study were primarily diuretics and/or digitalis derivatives. The clinical diagnosis of probable

Alzheimer’s disease was made conjointly by a neurologist and a psychiatrist on the basis of the insidious

onset of dementia with progression in the absence of other systemic or brain diseases that may cause

dementia. The age at onset of symptoms of dementia was determined to the nearest year according to

Biological/clinical subtype of Alzheimer’s disease 685

history provided by the patient, the patient’s family, and the treating physician. Duration of illness was

determined by subtraction of the age at onset from the patient’s age at the time of blood drawing. The

degree of cognitive impairment was graded by use of the Mini-Mental State score (O-worst; 30-best)

(Folstein et al 1975) and for Alzheimer’s Disease the Dementia Rating Scale (O-best; 2%worst) (Blessed et

al 1968).

A total of 24 elderly, depressed, nondemented inpatients were included as a comparison group. All

subjects in this group met Schedule for Affective Disorder and Schizophrenia, Lifetime Version and

Research Diagnostic Criteria for major depressive disorder (primary, unipolar, nondelusional) (Endicott

and Spitzer 1978), DSM-III criteria for major depression without psychotic features (APA 1980), and had

Hamilton Depression Rating Scale scores 215 (single rater, first 17 items) (Hamilton 1960). To exclude

patients with dementia, a Mini-Mental State score 226 also served as a criterion for inclusion in this group.

Patients in this group were medication-free for at least two weeks prior to blood drawing.

The clinical characteristics of the three diagnostic groups are presented in Table 1.

Table 1

Demographic and Clinical Characteristics of Subject Groups

Subject Group Mini-Mental Dementia Rating Hamilton Depression

M/F Age(y) State Score Scale Score Rating Scale Score

Healthy Controls n=50 18/32 66.7 19.4) 227 -__ <I5

Probable Alzheimer’s n=51 17134 68.6 (8.0) 16.8 (4.7) 7.3 (4.6) <I5

Depressed Controls n=24 8116 68 8 (7.4) 227 ___

Means are presented with standard deviations in parentheses. 23.6 (3.8)

Study Design

Blood drawing and blood cell isolations were performed according to a minor modification of the

method of Corash and coworkers (1977). A 20 ml fasting blood sample from each subject was drawn

between the hours of 9 a.m. and 12 noon by antecubital venipuncture through a 19 gauge needle into a

plastic syringe. Ten ml volumes were then transfened to each of two polypropylene plastic tubes (Falcon)

containing 25 mg of tetrasodium EDTA dihydrate and mixed by repeated inversion. In addition to its role

as an anticoagulant, EDTA served to inhibit potential proteolysis during processing. To ensure that all

blood processing and subsequent analyses would be carried out by personnel who were blind to

diagnosis, anticoagulated blood samples were coded by clinical staff before transport to the laboratory. All

platelet isolation procedures were conducted in plasticware at room temperature to minimize platelet

adherence to other blood cells as previously described (Zubenko et al 1987a-c). Total platelet membranes

were prepared from platelet suspensions as previously described (Zubenko et al 1987a-c). Red cell ghosts

were prepared as described by Dodge et al (1963).

686 G. S. Zubenko et al.

Platelet counts were determined with the use of a Model ZBI Coulter Counter equipped with a 50ptn

orifice and were in good agreement with counts determined by phase contrast microscopy. Final platelet

yields were >90% in all cases. Volume-frequency distributions were determined with a Cl000 Coulter

Channylzer calibrated with 2.02pm-diameter latex spheres,

For electron microscopy, platelets were fixed in pellets at 37OC for 30 min with 3% gluteraldehyde in

125 mM sodium cacodylate buffer with 7.5 mM sucrose at pH 7.3. Samples were rinsed at least 1 hr in

several changes of buffer then postfixed for 1 hr at room temperature with 1% osmium tetroxide in the

same buffer. Samples were dehydrated with ethanol and embedded in Epon araldite. Sections cut with a

diamond knife on a Sorvall Porter-Blum MT- 1 ultramicrotome were stained for 15 min with 3% aqueous

uranyl acetate and 10 min in Reynolds Lead Citrate then examined at 60 kV in a Philips 300 electron

microscope. Representative micrographs were taken of each coded platelet sample by an investigator who

was blind to clinical and laboratory data. Coded micrographs were then scored by the same individual for

abnormal platelets containing an overabundance of internal trabeculated cistemae bounded by smooth

membrane. Platelet preparations contained ~0.5% contamination by erythrocytes and leukocytes.

Intact platelets and cell membranes were diluted to a final optical density of 0.03 at 600 nm. Ten ml

suspensions of each were labeled by the addition of 10 ~1 of 1 mM solutions of DPH or TMADPH in

dimethylformamide. Labeling was carried out in the dark at final probe concentrations of 1 FM for 60 min

at 37OC (Shinitzky and Barenholz 1978, Kuhry et al 1985, Simons et al 1985, Zubenko et al 1987a-c).

Labeled membranes contained approximately 1 probe molecule to 100 phospholipid molecules.

Fluorescence measurements were performed on an SLM 4800 spectrofluorometer equipped as previously

described (Zubenko et al 1987a-c). All measurements were made at 37.0 f O.loC with stirring. Corrected

fluorescence spectra were obtained for each sample at 37.0 f O.loC and spectra characteristic of DPH and

TMADPH were observed in each case. For steady-state anisotropy measurements, the instrument was

configured in the T-format, labeled membranes were excited at 360 nm, and emission light was measured

through Schott long pass filters. Steady-state anisotropy measurements provide a reliable and valid index

of membrane “fluidity” or “order” over the range of values reported (Heyn 1979, Jahnig 1979, Van

Blitterswijk et al 1981, Pottell et al 1983). As a control, fluorescence lifetimes were measured by the

phase-modulation method (Spencer and Weber 1969) using excitation frequencies of 6, 18, or 30 MHz, an

excitation slit width of 0.5 nm, a solution of POPOP in ethanol as a lifetime standard (2=1.35nsec), and

with the emission polarizer oriented at 5.5O.

Because membrane preparations were stored and studied in PBS for fluorescence spectroscopy, they

required washing before the extraction and determination of phospholipids. One and one-half ml aliquots

of membrane suspensions in phosphate buffered saline were sonicated for 10 seconds at 35% power. The

suspension was diluted to 5 ml with 50 mM Tris-HCl, pH 7.4, and centrifuged in 5 ml polyallomer tubes

at 100,000 xg in a swinging bucket rotor. The supematant was discarded, and the pellet was resuspended

in 1.0 ml of the same buffer by sonicating on ice for 10 seconds. No phosphate contamination was

apparent after this procedure and over 90% of lipids, both cholesterol and phospholipid, in the original

sample were present in the washed suspension (Cohen et al 1987). Lipids were extracted by the procedure

of Bligh and Dyer (1959) as modified by Kawasaki et al (1984). Phospholipids were measured as total

Biological/clinical subtype of Alzheimer’s disease 687

lipid phosphorus by the method of Rouser et al (1970). Cholesterol was determined by the method of

Rude1 and Morris (1973). Protein was determined by the Biorad microassay with bovine gamma globulin

as a standard.

Data Analvsis

For continuous variables, comparisons of corresponding values between groups were made using one-

way analyses of variance with post hoc two-tailed t tests. Between group comparisons of categorical

variables were made using the chi-square statistic or Fisher’s exact test Relationships between continuous

variables were explored using the parametric Pearson correlation coefficient. All comparisons that

revealed statistically significant differences according to the t test remained significant when examined by

the non-parametric Mann-Whitney test. Likewise, those pairs of variables for which the Pearson

correlation coefficient was significantly different from zero were also related by a statistically significant

Spearman (non-parametric) correlation coefficient of the same sign.

Informed Consent

All subjects provided written informed consent prior to admission to the study. For demented patients,

written informed consent was also obtained from a family member.

Results

Platelet Membrane Fluidity in Alzheimer’s Di- and Deph

Analyses of membrane fluidity were performed at 37.00 + O.loC with the fluorescent probes DPH and

TMADPH, which label the membrane hydrocarbon and lipid-aqueous interface, respectively (Prendergast

et al 1981, Shinitzky and Barenholz, 1978). Steady-state anisotropies am presented in Figure 1. The

fluorescence anisotropy of DPH in labeled platelet membranes differed significantly among the diagnostic

groups as determined by a one-way analysis of variance (pcO.0001). The mean steady-state anisotropy of

DPH in platelet membranes from the demented group, 0.1920, was significantly decreased when

compared to the respective means for the healthy control group, 0.1991 (p=2.3 x 10-e), and the

depressed group, 0.1982 (p=7.8x10e4). The mean DPH anisotropy values for the healthy and depressed

control groups did not differ significantly from one another. Analysis of variance did not reveal a

significant effect of diagnostic group on the fluorescence lifetime of DPH in these labeled platelet

suspensions. Since DPH localizes preferentially within the hydrocarbon core of lipid membranes, these

results indicate that dementia of the Alzheimer-type is associated with an increase in the fluidity (inversely

related to anisotropy) of the hydrocarbon core of platelet membranes.

In contrast to the findings with DPH, analysis of variance did not reveal a significant effect of diagnostic

group on either the steady-state anisotropy or the fluorescence lifetime of TMADPH in labeled platelet

membranes from the study population. Since TMADPH is anchored to the lipid aqueous interface of

labeled membranes, these results imply a selective alteration in molecular dynamics at the hydrocarbon

region of platelet membranes from patients with Alzheimer-type dementia that does not extend superficially

to affect the lipid-aqueous interface at 37oC.

688 G. S. Zubenko et al.

0.22

0.21

0.18

0.17

0.16

8 0

8

HEA;THY PROLISLE DEPRk CONTROLS AD CONTROLS

Figure 1. Steady-state anisotropy values (370C) for DPH-labeled platelet membranes from subjects in the healthy control group (n=50), Alzheimer’s disease group (n=51), and non-demented, depressed group (n=24). Croup means marked by hori-.ontal bars were 0.1991 (S.D. 0.0058), 0.1920 (S.D. O.OOSO), and 0.1982 (S.D. 0.0048), respectively.

The temperature-dependence of DPH and TMADPH anisotropy between 5OC and 55oC was used to

determine whether platelet membranes from patients with Alzheimer’s disease had significant alterations in

flow activation energies (AR) at either the hydrocarbon core or the lipid aqueous interface region (Zubenko

et al 1987d). No significant changes were detected in either region (n=lO). Moreover, the phase

transition at the lipid-aqueous interface detected with TMADPH occurred at a mean temperature of 29.OoC

in both groups. These results imply that the observed increase in mobility of DPH in platelet membranes

from patients with Alzheimer’s disease is not the result of a gross alteration in the bioenergetics of

membrane lipid diffusion. Instead, these findings suggest a more specific alteration in platelet membranes

fluidity at the hydrocarbon core that persists over the range of temperatures studied.

Relationship of the 1 . . . .

PlateetMembraneAlterationfthes 1

The observed increase in platelet membrane fluidity, as reflected by a reduction in the steady-state

anisotropy of DPH-labeled membranes, was significantly correlated with clinical severity as measured by

either the Mini-Mental State score (Folstein et al 1975) or the Dementia Rating Scale score (Blessed et al

1968). The Mini-Mental State score and DPH anisotropy were best related by a reciprocal model of the

Biological/clinical subtype of Alzheimer’s disease 689

form l/Y=a+bX (Y=Mini-Mental State score, X=DPH anisotropy), with an association correlation

coefficient of -0.60 (l/Y vs. X, p=7.9xlO-5). Dementia Rating Scale score and DPH anisotropy were

best related by a linear model of the form Y=a+bX (Y=Dementia Rating Scale score, X=DPH anisotropy)

with an associated correlation coefficient of -0.64 (Y vs. X, p=1.6 x 10m4). The magnitude and statistical

significance of both correlations were increased by the inclusion of the most severely demented patient in

the group. However, even after the exclusion of this outlier, the correlations remained statistically

significant (although the respective correlation coefficients decreased in magnitude). Regardless of the

analysis performed, the degree of the observed alteration in platelet membrane fluidity paralleled dementia

severity as reflected by either the Mini-Mental State score or Dementia Rating Scale scores.

In contrast to clinical severity, neither age at onset nor duration of illness were significantly correlated

with the steady-state anisotropy of DPH in labeled platelet membranes from patients with Alzheimer-type

dementia. While we have previously reported a statistically significant positive correlation of the steady-

state anisotropy of DPH in labeled platelets with the age of healthy subjects from the second to the ninth

decade (Cohen and Zubenko 1985), the correlation of these variables within the healthy control group in

this study did not reach statistical significance. This apparent discrepancy most likely resulted from the

narrower range of the healthy controls employed here.

Analysis of variance revealed a trend for patient gender to affect the steady-state anisotropy of DPH in

labeled platelets within the study population. No significant effect of gender on the fluorescence lifetime

of DPH was observed in the total study population or within any of the three diagnostic groups. Finally,

as expected, a mean steady-state anisotropy of DPH-labeled platelet membranes from the 7 demented

patients who were taking medications that met our inclusion criteria did not significantly differ from that of

the 44 who had not been treated with medications at the time of entry into the study.

Characteristics of Demented Patients with “Normal” or “Increased’ Platelet Membrane Fluiditv

Inspection of the scattergram of DPH anisotropy values for labeled platelets from the patients with

Alzheimer-type dementia suggested a bimodal distribution with a nadir near the mean for the entire group,

0.1920. The lower 5th percentile of DPH anisotropy values among the controls (healthy and depressed

controls), corresponding to those <0.1920, was defined as “increased” platelet membrane fluidity. Since

the fluorescence anisotropy of DPH-labeled platelet membranes did not exhibit a significant correlation

with subject age above 45 years, age-correction of the proposed cutoff value was unnecessary. Twenty-

eight of the 51 demented patients (55%) fell within the increased fluidity subgroup, while (45%) fell

within the normal fluidity group. With 0.1920 as the cutoff value, only 4 of the 50 healthy controls (8%)

and none of the depressed controls had DPH anisotropy values outside of the “normal” range.

The characteristics of the demented patients in the subgroups with normal or increased platelet membrane

fluidity are presented in Table 2. The mean DPH anisotropy value for the normal fluidity subgroup was

very similar to the means for both the healthy and depressed control groups. The mean age of patients in

the increased fluidity subgroup, was significantly less than that of the normal fluidity subgroup. The sex

ratios of the subgroups, were similar to one another and to the sex ratio for the entire demented group.

690 G. S. Zubenko et al.

The mean age at onset of dementia for the patients in the increased fluidity subgroup, was significantly less

than that for the normal fluidity subgroup. The patients in the increased fluidity subgroup were also more

severely affected at the time of their entry into the study, as reflected by a significantly higher mean

Dementia Rating Scale score, than patients in the normal fluidity subgroup. Since the two subgroups did

not differ with respect to mean duration of illness, these data imply that the subgroup with increased

fluidity suffered a more rapid decline on average than did patients in the normal fluidity subgroup. In

summary, the subgroup of patients with Alzheimer’s-type dementia who manifest increased platelet

membrane fluidity (DPH anisotropy ~0.1920) were younger at the time of clinical onset, were more

severely demented, and suffered from a more rapid progression of their illness than those patients in the

normal fluidity group.

Table 2

Demographic and Clinical Characteristics of Demented Patients with “Normal” or “Increased Platelet Membrane Fluidity

Normal Fluidity Increased Fluidity (DPH Anisotropy 20.1920) (DPH <0.1920) P Value

DPH Anisotropy 0.1987 (0.0042) 0.1862 (0.0057) __.

Age (Y) 71.7 (8.2) 66.0 (7.1) 0.010

M/P 9114 8120 NS

Age at Onset 69.0 (8.7) 63.6 (5.9) 0.0028

Mini-Mental State Score 19.5 (4.3) 15.1 (4.1) 0.003

Dementia Rating Scale Score 3.8 (2.6) 9.6 (4.3) 0.0003

Duration of Illness 4.0 (2.3) 4.3 (2.1) NS

Means are presented along with standard deviations in parentheses. Comparisons of corresponding means were made using a two-tailed t test.

Pl 1 Mmr A * i nR fl 1 Cel lation Dvnami

We considered the possibility that the observed increase in platelet membrane fluidity associated with

Alzheimer’s disease resulted from a decrease in the mean age of circulating platelets in this pathologic

group. Since platelets decrease in volume with increasing cell age (Corash et al 1977), we determined the

mean volume of the platelets isolated from study subjects as a relative index of mean cell age. The mean

volume of platelets prepared from the demented group, 4.56 pm3 + S.D. 0.49 pm3, did not significantly

differ from the respective volume for the control group, 4.32 ym3 + S.D. 0.61 nm3. This observation

suggests that the increase in platelet membrane fluidity observed for the demented group did not result

from a reduction in the mean age of circulating platelets compared to the neurologically-healthy control

group.

Biological/clinical subtype of Alzheimer’s disease 691

As shown in Figure 2, the platelet specimens from the demented group contained frequent examples of

unusual cells that exhibited the proliferation of a system of trabeculated cisternae, bound by ribosome-free

membranes. This system did not appear to be continguous with the plasma membrane in any of the

photomicrographs and, therefore, seems unlikely to represent an overdevelopment of the cannalicular

system. Instead, this membrane system appears to lie internal to the platelets in which it is found. Blindly

rated, the mean percentage of these abnormal platelets in the Alzheimer’s disease group (n=6), 15.5% *

SD. 9.3%, was over three times greater than that observed for the age- and sex-matched group of

neurologically-healthy controls (n=6), 4.3% + S.D. 3.4% (p=2.2 x 10s2). In addition, the frequency of

abnormal platelets exhibited a significant negative correlation (r= -0.61, p=3.7 x 10-2) with the

fluorescence anisotropy of DPH-labeled platelet membranes prepared from the respective platelet

suspensions. Since internal platelet membranes have been reported to be more fluid than external platelet

membranes (Menashi et al 1981), the observed ultrastructural difference is consistent with the fluorescence

findings.

Figure 2. Transmission electron micrographs (22,000 x) of platelets from a neurologically-healthy control (left) and a demented patient with probable Alzheimer’s disease (right).

If the increase in platelet membrane fluidity observed for the Alzheimer’s group resulted from the

proliferation of a more fluid internal membrane compartment, then comparisons of intact platelets labeled

with DPH should reveal little, if any, difference in probe mobility between the demented and control

groups, since most probe would be dissolved in external membrane. Only a non-significant trend in the

direction of increased fluidity emerged in specimens from patients with probable Alzheimer’s disease

(Zubenko et al 1987c). In addition, this hypothesis predicts that red cells from patients in the demented

group would fail to exhibit an alteration in membrane fluidity, since they lack internal membranes. This

prediction was also realized (Zubenko et al 1987c). This result is also consistent with the report of

Markesbery and coworkers (1980) who employed ESR spectroscopy to examine red cell ghosts labeled

with 5nitroxide stearic acid and found no differences in membrane characteristics between patients with

Alzheimer’s disease and control subjects.

692 G. S. Zubenko et al

PI . . .ateletas.e

For biological membranes, the ratio of cholesterol to phospholipid is a major determinant of DPH

anisotropy (for reviews see Shinitzky and Barenholz 1978, Shinitzky and Henkart 1979, Cohen and

Zubenko 1985). In addition, internal cell membranes have ben reported to have a lower cholesterol to

phospholipid ratio than plasma membranes (Lagarde et al 1982, Fauvel et al 1986). Thus, to further

explore the abnormality of cell membranes observed in patients with Alzheimer’s disease, we have

determined the cholesterol and phospholipid content of platelet and erythrocyte membranes from our

subjects. The finding of a low cholesterol to phospholipid ratio in platelet membranes from our patients

with Alzheimer’s disease would both provide a possible biochemical explanation for the observed decrease

in DPH anisotropy and support the physical finding of an apparent increase in abundance of otherwise

normal internal membranes in these platelets.

Age and sex of the subjects and the results of determinations of membrane protein, cholesterol,

phospholipids, and the steady-state anisotropy of DPH are given in Tables 3 and 4, respectively, for

platelets and erythrccytes. DPH anisotropy was known to be low in platelets of patients with Alzheimer’s

disease. While cholesterol, per se, tended to be lower and phospholipids tended to be higher in platelets of

patients with Alzheimer’s disease, these differences did not approach significance. However, the ratio of

cholesterol to phospholipid was significantly lower (p<O.Ol) in platelets from patients with Alzheimer’s

disease than in the platelets of control subjects. Furthermore, the cholesterol to phospholipid ratio in

platelet membranes for all subjects and the steady-state anisotropy of DPH were significantly correlated

(r=0.53, p<O.Ol), suggesting that the two parameters are measuring different aspects of the same

phenomenon. Finally, the two groups were well matched for age, but it is worth noting that within the

limited age range of the subjects studied, neither cholesterol nor the ratio of cholesterol to phospholipid

correlated with age.

For erythrocytes, no significant differences nor any substantial trends towards a difference were

observed between the patient and control groups for any parameter. Furthermore, there was no correlation

between the cholesterol to phospholipid ratios observed in platelets and those observed in erythrocytes.

Erythrocytes had higher ratios of cholesterol to phospholipid (pdO.01) as well as higher steady-state

anisotropy of DPH (p<O.OOl) than platelets, a finding which is consistent with past observations that the

membrane cholesterol to phospholipid ratio is a major determinant of DPH anisotropy.

As did the results of fluorescence spectroscopy and electron microscopy, the finding of a low cholesterol

to phospholipid ratio in platelets suggests that membrane characteristics are abnormal in Alzheimer’s

disease. However, the findings do not suggest a general abnormality of cell membranes, since erythrocyte

membranes are normal both in cholesterol to phospholipid ratio and by fluorescence spectroscopy.

Similarly, the findings do not suggest that there are altered levels of cholesterol or phospholipids

themselves in serum or cell membranes of patients with Alzheimer’s disease since neither cholesterol nor

phospholipids per se were abnormal in amount in either platelets or erythrocytes of these patients.

Rather, since internal membranes are normally low in their ratio of cholesterol to phospholipid, the

findings support the results of ultrastructural studies which suggest that platelets of patients with

Biological/clinical subtype of Alzheimer’s disease 693

Table 3

Cholesterol, Phospholipid, and Anisotropy of DPH in Platelets of Patients with Alzheimer’s Disease and Control Subjects

Lipid Cholesterol Phosphorus (C) (P)

Protein Wmg ktg/mg DPH*** Group Pair Age Sex (mg/ml) protein protein c/P** Anisotropy

AD L I :

74 71 80

M F

: 5

!

8 10

62 64 65 58 58 70 73

F F F

; F

G

0.076 118 9.94 11.82 .1902 0.113 124 14.80 8.33 .1888 0.119 165 19.40 8.51 .1650 0.076 1.50 14.60 10.23 .1887 0.138 128 13.50 9.12 .1916 0.166 132 15.40 8.59 .1871 0.169 104 11.40 9.11 .1793 0.059 174 18.40 9.44 .1886 0.121 143 17.20 8.33 .1900 0.103 99 9.65 10.21 .1916

Mean 67.5 0.114 134 14.40 9.37 .1861 (SD) (7.3) (0.037) (24.5) (3.37) (1.11) (0.0083)

z s HE E A I s ME E R S

0.080 111 9.28 11.96 .1971 0.084 105 10.10 10.44 .1960 0.140 133 15.00 8.89 .2032 0.300 199 18.00 11.06 .2099 0.130 145 15.90 9.47 .1943 0.213 141 16.20 8.68 .1949 0.097 158 16.30 9.69 .I937 0.095 149 13.30 11.20 .1967 0.097 126 12.50 10.10 .I979 0.077 135 12.90 10.47 .1982

Mean 67.6 0.131 140 14.00 10.20 .1982 (SD) (7.9) (0.072) (26.3) (2.83) (1.04) (0.0049)

*Protein concentration after washing and resuspension of platelets prior to extraction of lipids. **Ratio of cholesterol as pg/mg protein to lipid phosphorus as pg/mg protein.

***Steady-state anisotropy of DPH dissolved in platelet membranes.

Alzheimer’s disease have a relative excess of internal membranes (Zubenko et al 1987~). The results

support the further speculation that the internal membranes themselves are not abnormal in their

composition. To explain all of the findings, by fluorescence spectroscopy, electron microscopy, and lipid

analysis, these membranes need only be present in overabundance.

Consideration of Snecificitv Discussion

These results confirm and extend our preliminary reports of a platelet membrane abnormality associated

with Alzheimer’s disease. An increase in platelet membrane fluidity, as reflected by a significant decrease

694 G. S. Zubenko et al.

Table 4

Cholesterol, Phospholipid, and Anisotropy of DPH in Erythrocytes of Patients with Alzheimer’s Disease and Control Subjects

Group

Lipid Cholesterol Phosphorus (C) (P)

Protein Fglmg pglmg DPH*** Pair Age Sex (mg/mI) protein protein c/P** Anisotropy

L”f fIE E A I s ME E R S

: N T R 0 L S

Mean (S.D)

74 M

:3 : 71 F

F :: F

F 5”: F 70 73 ;

67.1 (7.8)

M 56; F

70 80 FF 80 F 59

; ;; F 70 70 &

68.5 (7.6)

1.43 165 14.9 11.07 .2271 1.62 298 23.1 12.90 .2210 2.09 289 24.1 11.99 .2188 1.41 259 21.0 12.33 .2180 1.27 256 25.1 10.20 .2050 1.18 178 15.7 11.34 .2167 1.38 226 19.8 11.41 .2166 1.76 173 14.4 12.01 .2187 1.47 185 15.8 11.71 .2180 2.35 215 18.8 11.44 .2158

1.60 224 19.3 11.64 .2176 (0.37) (49.2) (3.99) (0.74) (0.0054)

0.92 239 18.5 12.92 .2250 1.72 161 13.8 11.66 .2128 1.39 182 15.9 11.45 .2255 1.28 198 18.9 10.48 .2190 1.23 260 21.6 12.03 .2214 1.98 176 15.9 11.07 .2144 1.71 253 26.4 9.58 .2219 1.75 208 16.1 12.91 .2156 1.36 290 23.0 12.61 .2210 1.94 254 19.7 12.89 .2152

1.53 221 19.0 11.76 .2192 (0.34) (42.9) (3.84) (1.14) (0.0045)

*Protein concentration after washing and resuspension of platelets prior to extraction of lipids. **Ratio of cholesterol as pg/mg protein to lipid phosphorus as pglmg protein.

***Steady-state anisotropy of DPH dissolved in platelet membranes.

in the steady-state anisotropy of DPH-labeled membranes, has now been reported by us for groups of

patients with Alzheimer’s disease in Boston and Pittsburgh (Zubenko et al 1984, Zubenko and Cohen

1985b, Zubenko et al 1987a-d), and by others for patients in London (Hicks et al 1987). Initial

assessments of the specificity of this platelet membrane parameter are promising. The increase in platelet

membrane fluidity associated with Alzheimer’s disease was not found in platelets from patients with

depression, a common cause of reversible dementia in the elderly (Post 1975), mania (Zubenko and Cohen

1985a), which may also be accompanied by a secondary dementia (Thase and Reynolds 1984), or multi-

infarct dementia (Hicks et al 1987). In addition, we have previously reported that platelet membrane

fluidity decreases from the second to the ninth decade in neurologically-healthy controls (Cohen and

Zubenko 1985). Therefore, our findings do not support the hypothesis that Alzheimer’s disease results

Biological/clinical subtype of Alzheimer’s disease 695

from a pathological acceleration of the normal aging process. The weight of the available evidence

suggests that the increase in platelet membrane fluidity associated with Alzheimer’s disease may be due to

a dysregulation of platelet membrane biogenesis or turnover that results in the accumulation of internal

platelet membranes.

Consideration of Possible Sources of Artif&

None of several possible sources of artifact considered could account for the observed increase in platelet

membrane fluidity associated with Alzheimer’s disease. Investigator bias was eliminated by ensuring that

laboratory staff remained blind to clinical data and, conversely, that clinicians remained blind to the

biophysical data. Fasting blood samples were employed in order to minimize the possible effects of

eating, even though we have found no effect of the ingestion of single meals on the platelet membrane

characteristics measured. While the isolation of different subpopulations of platelets from the comparison

populations was a potential source of bias, platelet yields for the samples were >90% in all cases. None of

the patients were receiving treatment with medications that affect platelet membrane fluidity, including

neuroleptics and antidepressants, and none of the demented patients or controls had any reported history

of exposure to such medications. Furthermore, it is unlikely that our findings would result from

unreported exposure to these medications based upon the effects that these agents have on the physical

properties of cell membranes. Phenothiazes increase the steady-state anisotropy of DPH-labeled platelet

membranes while haloperidol and tricyclic antidepressants have no effect on this platelet membrane

parameter at clinically relevant concentrations (Zubenko and Cohen, 1985a-c). Finally, the increase in

platelet membrane fluidity associated with Alzheimer’s disease does not appear to result from nonspecific

effects of chronic illness since 1) the steady-state anisotropy of DPH labeled platelet membranes from the

demented group was not significantly correlated with duration of illness and 2) none of the patients in the

study were suffering from malnutrition or vitamin deficiency syndromes that often accompany chronic

debilitation, and 3) depression in the elderly that was sufficiently disabling to warrant hospitalization was

not accompanied by an increase in platelet membrane fluidity.

Relationshin to Other Potential Subtvnes of Alzheimer’s Disw

Historically, dementia in the elderly has been separated into forms with presenile (~65~) or senile (?65y)

onset. This distinction was based largely on the clinical observation that patients who developed

symptoms of dementia at an early age often had a more rapid decline and more often had family members

with dementia than those whose symptoms developed later in life. In our study, the demented subgroup

with increased platelet membrane fluidity bears a resemblance to the syndrome presenile dementia, made

on clinical grounds. These patients exhibited an earlier onset of symptoms, were more severely demented

as measured by both the Mini-Mental State and Dementia Rating Score scales, and suffered a more rapid

deterioration than patients with “normal” platelet membrane fluidity. Therefore, the platelet membrane

abnormality appears to describe a clinically distinct subtype of patients with Alzheimer’s disease.

Population and family studies have uniformly found that first-degree relatives of patients with

Alzheimer’s disease are at a significantly higher lifetime risk of developing dementia, especially if the

696 G. S. Zubenko et al

affected family member is a parent and the onset occurs before age 70 @amson et al 1963, Heston et al

1981, Heyman et al 1983, Breitner and Folstein 1984, Silverman et al 1986). Most recently, family

studies have revealed that the increase in platelet membrane fluidity associated with Alzheimer’s disease is

a familial trait that aggregates in neurologically-healthy fmt degree relatives of probands with Alzheimer’s

disease (Zubenko et al, unpublished results). This finding strongly suggests that the platelet abnormality

is related to, but antedates, the onset of symptoms of Alzheimer’s disease. This results also excludes

nonspecific concornmitants of chronic illness or medication history as the cause of the increased platelet

membrane fluidity observed. Furthermore, the aggregation pattern of increased platelet membrane fluidity

within the high-risk cohort was consistent with that expected to result from the expression of a fully-

penetrant autosomal dominant gene. In summary, these family studies suggest that increased platelet

membrane fluidity is a genetic marker for familial Alzheimer’s disease and that the platelet membrane

abnormality is inherited as an autosomal dominant trait.

Conclusion

Increased platelet membrane fluidity identifies a subgroup of about 50% of patients with Alzheimer’s

disease that is associated with distinct clinical features, including early onset and a rapidly progressive

course. The weight of the available evidence suggests that this membrane abnormality may result from a

dysregulation in the biogenesis or turnover of internal membranes. This abnormal membrane characteristic

is familial and the aggregation pattern is consistent with the inheritance of a fully-penetrant autosomal

dominant trait.

Acknowledoement

This work was supported by Alzheimer’s Disease Research Center grant AGO5133 (GSZ and FB) and

Mental Health Clinical Research Center grant MH3091510 (GSZ); project grants MH38313 (BMC) and

MH37869 (CFR); and program grants AGO3705 (GSZ and FB), MH36224 (BMC), and MH31154

(BMC). GSZ and CFR were recipients of NIMH Research Scientist Development Awards MHOO540 and

MHoo295.

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Inquiries and reprint requests should be addressed to:

George S. Zubenko, M.D., Ph.D. Western Psychiatric Institute and Clinic 3811 O’Hara Street, Room E-1231 Pittsburgh, PA 15213 U.S.A.